1. Field of the Invention
The present invention relates to a novel recombinant (rec-) L-N-carbamoylase from Arthrobacter aurescens as well as to a method of producing L-amino acids with this carbamoylase.
The L-amino acids which can be advantageously produced with the novel rec-carbamoylase are important chiral starting materials for the organic synthesis, for example, of drugs and have significance in human and animal nourishment.
2. Background Information
The advantageous possibility of the stereoselective splitting of the D,L-carbamoyl amino acids assessable from the corresponding hydantoins by hydantoinases by means of L-N- and/or D-N-carbamoylases resulted previously in the establishment of methods for the production of L- and D-amino acids on an industrial scale. L-specific carbamoylases have been detected in very different strains of microorganisms (Ogawa et al., J. Mol. Cal. B: Enzym. 1997, 2, 163-176; Syldatk et al. in Drauz, K. and Waldmann, H.: Enzyme Catalysis in Organic Synthesis, Weinheim: VCH-Verlag, 1995).
The presence of L-N-carbamoylase activity in cells of the strain Arthrobacter aurescens has been known for some time. This carbamoylase has been isolated in homogeneous form and the N-terminal sequence has been determined. (Müller, dissertation, Braunschweig TU [Technical University], 1990). However, the gene coding for the enzyme was unknown and has therefore not been previously cloned or overexpressed. Thus, the recombinant enzyme and its amino acid sequence and the DNA coding the amino acid sequence were previously unknown.
Three L-carbamoylases produced in a recombinant manner have been described in the literature (Batisse et al., Appl. Environ. Microbiol. 1997, 63, 763-766; Mukohara et al., Biosci. Biotechn. Biochem. 1993, 57, 1935-1937; Watabe et al., J. Bacteriology 1992, 174, 962-969). However, the L-carbamoylases cited there are relatively unstable and are thus unsuitable for being used in an industrial process.
The L-specific carbamoylase from Arthrobacter aurescens (A.a.) was able to be used previously for the splitting of N-carbamoyl amino acids to free L-amino acids only as a predominantly homogeneously purified enzyme or in the form of free or immobilized, dormant [resting] whole cells. Several basic problems occur in the latter method. They include, among other things, transport limitations (especially for the N-carbamoyl amino acids), potential side reactions (degradation of the formed amino acids by other enzymes present) as well as contaminants in the product caused by lysis of the whole cells used, which make expensive purification processes necessary.
The L-carbamoylase from A.a. is very unstable in both forms used, which can be traced in the instance where dormant, immobilized cells are used to the natural protein turnover of the enzyme (Siemann, dissertation, Braunschweig TU, 1992). When used as a homogeneously purified enzyme, the lability is obviously affected by unavoidable changes in the protein concentration and the salt content. Moreover, the sensitivity of the wild enzyme to oxidation has been observed. In addition, only a very low yield of 2.7% is achieved in the purification of carbamoylase from the wild strain (Müller dissertation, Braunschweig TU, 1990). This low yield, the necessary complicated purification method, and the previously immanent lability of the enzyme prevent the use of this procedure in a competitive industrial method for obtaining L-amino acids.
The present invention therefore had the objective of obtaining an L-carbamoylase which is more stable than the L-carbamoylases of the prior art, and those previously producible from Arthrobacter aurescens, in a simpler form and with a better yield. However, the further requirements which a method places on the enzyme used on an industrial scale such as, for example, activity or selectivity regarding substrate and stereochemistry, etc. should not be negatively affected.
These and other problems not cited in detail but which will be evident to those of skill in the art are solved by rec-L-N-carbamoylase from Arthrobacter aurescens and mutants thereof, which are included in the invention. Their amino acid sequences and gene sequences derived therefrom are also included in the invention, as well as vectors modified with the gene sequences, corresponding plasmids, modified host organisms and cells of the host organisms.
In addition, the invention includes an advantageous method of producing L-amino acids using these enzymes of the invention.
As a result of the fact that L-N-carbamoylase is produced in a recombinant manner from Arthrobacter aurescens or its mutants, L-carbamoylases are obtained in very good yields of >90% and with a decidedly high purity which hare a much greater stability over the previously known carbamoylases, yet have a good activity and very good stereoselectivity as concerns the regarded biotransformation. These characteristics are essential for the successful use of carbamoylases in an industrial method for the production of L-amino acids.
It resulted in stability studies that homogeneously purified carbamoylase has only a few minutes activity at 50° C. (Müller dissertation, Braunschweig TU, 1990), whereas on the other hand rec-carbamoylase is active for hours. At 37° C. the activity of rec-carbamoylase is almost unchanged for 100 h (FIG. 2). This could in no way have been foreseen and is nevertheless all the more advantageous.
In addition thereto, the use of these recombinant carbamoylases opens up the possibility for the first time of obtaining industrial access to β-aryl-substituted L-amino acids by means of an enzymatic biotransformation via (D,L)-N-carbamoyl amino acids. The regarded carbamoylase is the only one of the previously known carbamoylases to also offer, in addition to the high L-enantioselectivity, the possibility of reacting [converting] β-aryl-substituted L-N-carbamoyl amino acids to an extent sufficient for an industrial method. (D,L)-formyl amino acids of this provenance are also suitable for being reacted with the enzymes in accordance with the invention. The free L-amino acid is also obtained with preference from the latter [formyl amino acids].
The amino acid sequences characterizing the recombinant L-N-carbamoylase from Arthrobacter aurescens and its mutants only have an agreement of maximally 38% with amino acid sequences of L-N-carbamoylases known from the state of the art. The novel and inventive gene sequences coding these amino acid sequences can be produced according to known biochemical methods. The vectors, plasmids and cells of host organisms containing these gene sequences are also novel and inventive. Preferred plasmids are pAW 16 and pAW 178-2 (FIG. 1), a preferred vector is pJOE 2702. Host cells can be in principle all microorganisms known to the expert in the art and coming into consideration for this purpose; however, E. coli JM 109 or E. coli W3110 is preferred.
The induction of the expression can be achieved in principle with all methods known to the expert in the art. However, rhamnose-, IPTG- and lactose systems are preferred.
The isolation and expression of the L-N-carbamoylase gene hyuC from Arthrobacter aurescens takes place as follows.
A gene bank representative for the entire genome of Arthrobacter aurescens and in an E. coli λRESIII vector was prepared and screened with an oligonucleotide which was derived from the N-terminal amino acid sequence of the purified hydantoinase from Arthrobacter aurescens. Plasmid pAW16 was obtained thereby which contained a 7.6 kb DNA fragment of Arthrobacter aurescens. Its nucleotide sequence was completely determined. The hydantoinase gene was identified from the nucleotide sequence. Another reading frame was identified on the C-terminal end of the hydantoinase gene, the derived amino acid sequence of which reading frame exhibited homology to an N-L-carbamoylase from Pseudomonas sp. and Bacillus stearothermophilus. Moreover, the start of the derived protein exhibited agreement with the N-terminal end, which was determined by amino acid sequencing, of the L-N-carbamoylase purified from Arthrobacter aurescens. The carbamoylase gene was amplified using PCR from pAW16 and inserted into an E. coli expression vector. Carbamoylase can be produced in the recombinant cells in a high amount via a promoter which can be induced by the sugar rhamnose.
SEQ ID NO:1 and SEQ ID NO:2 set forth the nucleotide and amino acid sequences, respectively, of the rec-carbamoylase from A.a. 
The term “mutants of L-carbamoylase” will be understood by the skilled artisan to mean enzymes which are produced by the exchange of individual amino acids, if necessary, but are derived from the rec-carbamoylase from A.a., and which conform to the characteristics outlined above, for use in the described method in a manner. Such mutants will be at least as good and possibly better (for workup, lesser sensitivity to oxidation, etc.). The term “mutants” also denotes such enzymes which are produced by the addition of amino acids or amino acid sequences to the rec-enzyme. Those are preferred which exhibit an His-TAG or Asp-TAG modification on the C-terminus.
The desired L-amino acids are produced in accordance with the invention with these advantageous L-N-carbamoylases from the corresponding L-N-carbamoyl amino acids and L-N-formyl amino acids. As has already been stated, the claimed L-N-carbamoylases exhibit a preferred reaction of β-aryl-substituted L-N-carbamoyl- and L-N-formyl amino acids. These chiral compounds can be produced from chiral hydantoins preferably by hydantoinases. The additional use of a chemical or enzymatic method for the racemization of hydantoins makes it possible in an extremely advantageous manner to construct an industrially valuable process for the obtention of L-amino acids from readily producible hydantoins. Chiral 5-monosubstituted hydantoins with aromatic side chains are to be considered with preference thereby. The stereoisomers of these hydantoins are constantly equilibrated by the established racemization step—if necessary, by a racemase—whereas the hydantoinase brings forth the corresponding L- or D,L-N-carbamoyl amino acids. The stereospecific rec-L-N-carbamoylases of the invention then assure the last step for the production of the L-amino acid since the hydantoinase from A.a. exhibits no absolute stereospecificity, in contrast to the carbamoylase. The preceding constant equilibration of the stereoisomers of the hydantoins assures that all hydantoin, whether D- or L-configured, is finally converted to an L-amino acid.
The protected amino acids are preferably reacted with the enzymes in a so-called enzyme membrane reactor. The stability of the enzymes used assures that the enzymes retained by an ultrafiltration membrane in the reactor can be used several times (>10 times) in succession for the production of L-amino acid. This saves expense and complexity for the method since only substrate must be charged and product drawn off.
Alternatively, the enzyme can be immobilized on carriers. This was successful in sufficiently high yield after the rec-enzyme was able to be used in pure form. All methods (covalent, adsorptive, etc.) known to the expert in the art for the immobilization of enzymes on carrier materials (silica gels, SiO2, EAH sepharose, nitrocellulose, Eupergit®) can be used; however, the covalent coupling to EAH sepharose obtained by carbodiimide is advantageous. When modified in this manner the enzyme can also be used in a fixed-bed reactor.
The following examples are intended to explain the invention without, however, limiting it in any manner.